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Complex disease genetics: Utilising targeted sequencing and homogeneous ancestry
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The complex disease investigations presented in this thesis aimed to provide new information regarding underlying genetics by using targeted sequencing and ethnically homogeneous cohorts. This work moved past current methodologies and addressed data stratification issues, that might have been hindering new findings. The results contribute to a more comprehensive view of the genetics of ankylosing spondylitis (AS) and breast cancer (BC), in Sweden.

Paper-I presents a sex-stratified analysis of a Swedish AS cohort that incorporated both common and rare variants. Single variant and aggregate tests both showed different signals in AS male and female patients, previously masked. Specifically, the RUNX3 locus in males (univariate test: rs7414934, OR=2.58, p=1.7x10-5) and MICB in females (SKAT: 27 variants, p=1.2x10-6; rs3828903, OR=4.62, p=6.2x10-13) exceeded discovery thresholds. In the functional follow up of these loci, risk alleles appear to regulate the expression of genes in multiple tissues. Also, the results highlight the importance of disease regulation from different haplotypes and loci breakdown proved that Sweden’s genetic architecture might be critical for AS studies.

Paper-II is a replication study, in our modest-sized Swedish cohort, of AS associations, previously discovered in populations of British origin, Initially, power calculations assessed that the Swedish cohort had the power to replicate only published associated markers with high effect (OR > 7), e.g., HLA-B but the replication analysis revealed three associated loci (ORrange:1.9-2.7). Notably, the multiplicated HLA-B marker (rs4349859) was not in HWE equilibrium. Population structure differences could not explain this replication pattern. However, sequencing resolution revealed fine-scale differences with repositioned association signals in the known loci. Specifically, the identification of two CCHCR1 protective haplotypes (OR: 0.14/0.3) that affect other MHC gene expression through eQTLs, provided the first suggestion of the differential function of known associated loci with cis gene regulation.

Paper-III provides the first fingerprint of the somatic mutation profile of Swedish BC. The significantly mutated genes were PIK3CA (28%), TP53 (21%) and CDH1 (16%) while histone-modifying genes (e.g., KMT2C and ARID1A: together 28%) exhibited an increased somatic mutation prevalence, not observed previously. Additionally, within the patients that did not receive neoadjuvant treatment, there were distinct age groups with different mutational profiles and differential APOBEC signature driving genes.

Taken together, these studies emphasize the contribution to the underlying genetics deriving from smaller ethnic populations, when assessed with a shift in methodology to account for biological bias, like sex and age. The results will hopefully assist and guide other genetic studies of human complex disease.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. , p. 62
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1588
Keywords [en]
ankylosing spondylitis, breast cancer, targeted sequencing, Sweden, genetics, population stratification.
National Category
Genetics
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:uu:diva-390457ISBN: 978-91-513-0710-7 (print)OAI: oai:DiVA.org:uu-390457DiVA, id: diva2:1341722
Public defence
2019-09-27, Room B22, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Note

Exact room will be known the 12th August. Booking system was down due to summer vacation

Available from: 2019-09-04 Created: 2019-08-10 Last updated: 2019-09-17
List of papers
1. The sex-stratified genetic architecture of ankylosing spondylitis
Open this publication in new window or tab >>The sex-stratified genetic architecture of ankylosing spondylitis
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Sexual dimorphism is an emerging feature of ankylosing spondylitis (AS), a chronic rheumatic condition affecting up

to three times more men than women. Using 691 individuals from a Swedish case-control cohort, we revealed that

sex biases are also a hallmark of AS genetic predisposition, and that this multifactorial disease is in part driven by

both rare and common variants. We identified SNPs via the targeted re-sequencing of 7 270 coding and non-coding

loci, and assessed novel patterns of association with both single marker and aggregate loci SKAT tests. The male

specific RUNX3 locus (including rs7414934, OR=2.58, p=1.7x10-5) and female specific MICB SKAT locus (27

variants, p=1.2x10-6; rs3828903, OR=4.62, p=6.2x10-13) exceeded discovery thresholds. Multiple risk variants from

each locus were shown to be functionally active in immune (Jurkat), skin (HaCat) and bone (SaOS-2) cell lines.

Differential patterns of genetic predisposition may point to alternative disease mechanisms in male and female

patients. Genetic and functional analyses demonstrated that risk alleles should not be considered in isolation and that

associated variants would likely affect gene regulation across multiple tissues. This work illustrates the need to

consider the contribution of sex to the genetics of AS and the duality that individual loci may play in the key clinical outcomes of disease.

Keywords
ankylosing spondylitis, SKAT, RUNX3, MICB
National Category
Genetics
Research subject
Molecular Genetics; Molecular Genetics
Identifiers
urn:nbn:se:uu:diva-390455 (URN)
Available from: 2019-08-10 Created: 2019-08-10 Last updated: 2019-09-24
2. Replication and fine mapping of ankylosing spondylitis replicated loci in the Swedish population reveal different CCHCR1 protective haplotypes
Open this publication in new window or tab >>Replication and fine mapping of ankylosing spondylitis replicated loci in the Swedish population reveal different CCHCR1 protective haplotypes
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: The genetics of ankylosing spondylitis (AS) derives mainly from studies performed in large cohorts of British origin. However, within Europe, disease prevalence is higher in Sweden, and so we investigated the reproducibility of known AS susceptibility patterns in a homogeneous Swedish cohort.

Methods: The replication power of the Swedish cohort was examined and a set of published SNP associations intersected with genotypes from an existing targeted sequencing study using these individuals (381 controls; 310 AS cases). To elucidate whether replication patterns derived from population subsampling or genetic similarity, allele frequency data from additional British and Swedish control populations were examined for genetic differentiation (FST). Replicated loci were fine mapped to investigate associations in more detail, and signals were dissected with haplotype analysis and functional annotation.

Results: The study had 80% power to find variants of strong effect (Odds ratio, OR>2) given a wide range of risk allele frequencies (0.2<RAF<0.8). From a set of 40 markers (33 gene loci), four variants were replicated (p-value < 1.25 x10-3), tagging HLA-B,CCHCR1and IL23R. The replication pattern was not due to European population genetic distance and fine mapping revealed genome-wide repositioned associations in HLA-Band CCHCR1, independent from the published associations (p-value < 2 x10-8, r< 0.3). The CCHCR1 locus showed two protective haplotype blocks (B1-1 and B2-1), independent from HLA-B signals (B1-1: r= 0.39, B2-2:r2=0.07), where 74% of controls were carrying 2 copies of the protective haplotypes (B1-1 and B2-1: OR=0.3, p-value = 1.2 x 10-45). Interestingly, while both haplotypes span CCHCR1, the effect of each haplotype is likely in cis, with eQTL evidence pointing to the regulation of TCF19(B1-1) and POU5F1(B1-2).

Conclusions: Both European populations share key disease loci, but the Swedish cohort revealed fine-scale genetic differences, that may point to gene regulation. This study utilized a different variant resolution, and by doing so demonstrated that smaller populations have the potential to reveal new AS pathogenesis mechanisms and that further study of the Swedish population is warranted.

Keywords
Ankylosing spondylitis, Swedish population, HLA-B27, CCHCR1, TCF19, POU5F1.
National Category
Genetics
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:uu:diva-390452 (URN)
Note

Please do not publish online in DiVa before publication: manuscript under review

Available from: 2019-08-10 Created: 2019-08-10 Last updated: 2019-08-10
3. Targeted sequencing reveals the somatic mutation landscape in a Swedish breast cancer cohort.
Open this publication in new window or tab >>Targeted sequencing reveals the somatic mutation landscape in a Swedish breast cancer cohort.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

 Background: Breast cancer (BC) is a genetically heterogeneous disease with high prevalence in

Northern Europe. However, there has been no detailed investigation into the Scandinavian somatic

landscape. Here, in a homogeneous Swedish cohort, we describe the somatic events underlying BC,

leveraging a targeted next-generation sequencing (NGS) approach.

 Methods: We designed a 20.5 Mb array targeting coding and regulatory regions of genes with a known

role in BC (n = 765). The selected genes were either from human BC studies (n = 294) or from within

canine mammary tumor (CMT) associated regions (n = 471). A set of predominantly estrogen receptor

positive tumors (ER+: 85%) and their normal tissue counterparts (n = 61) were sequenced to ~140x

and 85x mean target coverage, respectively. MuTect2 and VarScan2 were employed to detect single

nucleotide variants (SNVs) and copy number aberrations (CNAs), while MutSigCV (SNVs) and GISTIC

(CNAs) algorithms estimated the significance of recurrent somatic events.

 Results: The significantly mutated genes (q ≤ 0.01) were PIK3CA (28% of patients), TP53 (21%) and

CDH1 (11%). However, histone modifying genes contained the largest number of variants (KMT2C and

ARID1A, together 28%). Mutations in KMT2C were mutually exclusive with PI3KCA mutations (p ≤ 0.

001) and half of these affect the formation of a functional PHD domain. The tumor suppressor CDK10

was deleted in 80% of the cohort while the oncogene MDM4 was amplified. Mutational signature

analyses pointed towards APOBEC deaminase activity (COSMIC signature 2) and DNA mismatch

repair (COSMIC signature 6). We noticed two significantly distinct patterns related to patient age; TP53

being more mutated in the younger group (29% vs 9% of patients) and CDH23 mutations were absent

from the older group.

 Conclusions: The increased somatic mutation prevalence in the histone modifying genes KMT2C and

ARID1A distinguishes the Swedish cohort from previous studies. KMT2C regulates enhancer activation

and assists tumor proliferation in a hormone-rich environment, possibly pointing to a role in ER+ BC,

especially in older cases. Finally, age of onset appears to affect the mutational landscape suggesting

that a larger age-diverse population incorporating more molecular subtypes should be studied to

elucidate the underlying mechanisms.

Keywords
Breast cancer, PIK3CA, KMT2C, TP53, Sweden, targeted sequencing, NGS
National Category
Cancer and Oncology
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:uu:diva-390456 (URN)
Available from: 2019-08-10 Created: 2019-08-10 Last updated: 2019-08-15Bibliographically approved

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